J. Agric. Food Chem. 1002, 40, 1422-1424
1422
Electrophoretic Analysis of Cationic Proteins Extracted from Aflatoxin-Resistant/SusceptibleVarieties of Corn Joseph N. Neucere Southern Regional Research Center, Agricultural Research Service, U.S.Department of Agriculture, P.O.Box 19687, New Orleans, Louisiana 70124
The cationic proteins from two varieties of corn, Zea mays L., that are resistant (Yellow Creole) and susceptible (Huffman) to aflatoxin contamination were characterized by electrophoresis. Under native conditions, cathodic electrophoresis showed a cluster of proteins at Rf = 5.0-6.5 in Yellow Creole that was not evident in Huffman. SDS-PAGE showed approximately 12 bands that ranged between 14 and 43 kDa in both varieties. The profiles were different, both qualitatively and quantitatively. The major proteins in Yellow Creole clustered around 20 kDa on the gel and those in Huffman around 25 kDa. Qualitative variations of proteins by two-dimensional electrophoresis were evident, especially within the pH range 6.7-8.0 and around 27 and 92 kDa. The results are discussed in connection with the possible role of polypeptides as antifungal agents in species of corn.
In a previous study (Neucere and Zeringue, 19871, we showed that a large number of isocratic protein fractions recovered from anion-exchange cellulose chromatography from both Aspergillus f l a w s resistant and susceptible varieites of corn, Zea mays L., inhibited germination of fungal spores. Destructive activities on microbial cells by complex proteins associated with carbohydrates and lipids from both plant and animal tissues have been reported extensively in recent years (Albersheim and AndersonProuty, 1975; Barkai-Colan et al., 1978; Coleman and Robert, 1982; Olson et al. 1977; Roberts and Selitrennikoff, 1986). Many of these defense compounds exert lethal effects on biologicalsystems by destroying enzyme activity, distorting cell membranes, and other unknown mechanisms of interactions. This paper describes electrophoretic profiles of the cationic proteins from highly resistant (Yellow Creole) and highly susceptible (Huffman-white) genotypes of corn to A. flauus infection that were investigated earlier (Neucere and Zeringue, 1987; Zuber et al., 1983). MATERIALS AND METHODS Protein Extraction and Ion-Exchange Chromatography. Three hundred gram kernal samples of each variety were milled on a standard Wiley mill with a 40-mesh screen. Each meal was then extracted with 4 L of hexane to remove lipids and then subsequently air-dried. Twenty-gramsamples of the dried meals were extracted accordingto the method of Landry and Moureaux (1980)with three 200-mL portions of phosphate buffer in saline (0.9 mM NaHzP04.H~0,9.6mM NazHP04,0.5 M NaC1, pH 7.8) on a Tekmar homogenizer at 25 "C. The homogenate8 were clarified by centrifugation at l8000g for 30 min at 25 "C. The final supernants (550 mL each) were dialyzed in 3.5-kDa cutoff bags against three 8-L volumes of deionized water for 3 days in a cold room. The contents in each dialysis bag were freeze-dried and stored at -20 "C. For preparative chromatography, 80 mg of protein from each of the two varieties was dissolvedinto 250 mL of phosphate buffer, pH 7.8,0.03 ionic strength, dialyzed in the same buffer overnight, and absorbed on 4 g of a DEAE-cellulose(Bio-Rad cellex D) bed as described earlier (Neucere and Zeringue, 1987). In these experiments at least one-third of the protein applied was eluted in the isocratic fractions (tubes 1-58) which showed sporadic inhibition of A. flauus mycelial growth. These are the pooled fractions that were characterized in the current study. Analytical Procedures. Separation of native cationic proteins by cathodic polyacrylamidegel electrophoresis,PAGE (BioThis article not subject to U S . Copyright.
Rad), was conducted on vertical slabs at pH 8.5 according to the procedure of Mikola (1965) using a borate-potassium hydroxide buffer system. The stacking gels contained 4% (w/v) acrylamide, and the running gels contained 7.5% (w/v) acrylamide. Electrophoresis was conducted at constant current of 50 mA/ slab for approximately 5 h at 10 OC. SDS-PAGE was conducted according to the method of Laemmli (1970). The gels contained 5% (w/v) acrylamide in the stacking medium and 12% (w/v) acrylamide in the running gel. Bio-Rad markers ranging from 14.0 to 92.5 kDa were employed. The electrophoretic conditions were the same as for cathodic PAGE with reversed polarity. Densitometric tracings for all experiments were made with a (28-930 Shimadzu scanner at wavelengthof 600 mm. All gelswere stained with 0.125% Coomassie BlueR (Sigma)in methanol-aceticacidwater (4:2:5 v/v/v) and destained with the same solvent system. Two-dimensionalelectrophoresis was performed according to the method of O'Farrell (1975) by the Kendrick Laboratory (Madison, WI) as follows: Isoelectric focusing using 1.5% pH 5-7, 1-5% pH 5-8, and 1.0% pH 3.5-10 ampholines (LKB Instrumenta, Baltimore, MD) was carried out for 9600 V h (700 V for 13h 45 min). Forty nanograms of an IEF internal standard, vitamin D dependent calcium binding protein, MW 27 OOO and pZ = 5.2, was added to the samples. This standard is indicated by an arrow (M) on the stained 2-D gel. The final tube gel pH gradient extended from pH 4.1 to pH 8.1 as measured by a surface pH electrode (Bio-Rad) and colored acetylated cytochrome pZ markers (Calbiochem-Behring,La Jolla, CA) run in an adjacent tube. The following proteins (Sigma Chemical Co., St. Louis, MO) were added as molecular weight standards to the agarose which sealed the tube gels to the slab gels: myosin (220 OOO), phosphorylase A (94 OOO), catalase (60 OOO), actin (43 OOO), and lysozyme (14 OOO). These standards appear as fine horizontal lines on the silver stained 10% acrylamide slab gels (Oakley et al., 1980). The silver-stained gels were placed in 8% glycerol and transparency dried. Protein concentrations were determined according to either the BCA method (Pierce) using ovalbumin (Sigma) as standard or the micro-Kjeldahl technique. RESULTS AND DISCUSSION One-dimensional electrophoretic profiles and semiquantitation of the native cationic protein bands by densitometry are described in Figure 1. The densitometric tracings showed differences in relative concentration and migration of individual bands as recorded in Table I. The data showed t h a t a large proportion of total proteins was associatedwith t h e leading front of electrophoresis (bands 12 in A and B) from both Yellow Creole and Huffman. In
Published 1992 by the American Chemical Society
J. Agric. Food Chem., Vol. 40, No. 8, 1992
AflatoxlnReslstant/Susceptlb~Corn
1429
11
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Figure 1. Cathodic PAGE of native cationic proteins from two genotypes of corn [A, Yellow Creole (resistant); B, Huffman (susceptible)]. Densitometric traces of the gels were stained with Coomassie Blue R. Each sample slot was filled with 50 pg of protein. The origin of migration corresponds to the start of the separating gel, and the last peak in each tracing corresponds to the leading edge of electrophoresis. The relative concentration of each peak is described in Table I. The absorbance values (ordinates) for each genotype are staggered to enhance visualization of the tracings. Table I. SemiquantitativeComparison of Native Cationic Proteins in Yellow Creole (Resistant) (A) and Huffman (Susceptible) (B)Corn from Gel Tracings in Figure 1 migration, cm % area peak A B A B A B 1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
0.8 1.5 1.7 2.1 3.1 3.8 4.3 5.2 5.4 5.6 6.0 6.8
0.3 1.1 1.7 2.2 2.7 3.2 4.0 4.6 5.3 6.0 6.8
0.6 7.2 4.2 2.6 11.0 7.0 12.0 8.5 9.5 8.0 8.2 33.0
3.6 2.2 0.5 0.7 10.6 6.5 11.9 13.0 19.0 4.0 26.0
particular, the series of bands a t CM 5.0-6.5 in Yellow Creole (A) are distinct from those in Huffman (B)in that area of migration. Furthermore, peaks 9 and 10 in Yellow Creole appear as unresolved peak 9 in Huffman. T h e first seven peaks in the two varieties showed both slight (a) differences in electrophoretic migration and (b) relative concentration variations. Assessment of molecular weights by SDS-PAGE of the dissociated proteins from the two varieties and relative concentrations of individual bands is shown in Figure 2 and Table 11. For both varieties molecular size ranged between 11 and 42 kDa. A major peak of 26.5 kDa in Huffman (peak 5 in Figure 2,trace B)a t CM 6.5was not observed in large quantity in Yellow Creole. In addition, some differences were observed in the two profiles a t CM
Figure 2. SDS-PAGE of the cationic proteins in the two corn genotypes [A, Yellow Creole (resistant);B, Huffman (susceptible)]. Densitometric scans of gel patterns start at the origin of the separating gel. Molecular weight markers (20 pg) ranging from 14 OOO to 92 500 were used to establish a standard curve. The last peak in each tracing corresponds to the leading edge of electropheresis. Each test sample slot was loaded with 50 pg of protein along with Bromophenol Blue as a tracking dye. Relative concentration of each peak and estimated molecular weight are given in Table 11. Staggered absorbance values enhance comparison of tracings. Table 11. Semiquantitation and Molecular Weight Determination of Dissociated Cationic Proteins in Yellow Creole (Resistant) (A) and Huffman (Susceptible) (B)Corn from Gel Tracings in Figure 2 molecular weight peak 5% area A B A B A B 1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
1.6 4.9 2.8 4.6 7.2 8.6 3.1 4.4 14.1 13.2 12.9 3.0
3.5 1.2 1.5 4.5 34.1 1.2 11.4 1.0 7.6 17.5 2.7
44 OOO 30 OOO 24 500 22 OOO 20 500 19 500 18 500 17 500 16 OOO 14 OOO 13 OOO 11 500
41 500 37 OOO 32 OOO 29 500 26 500 21 500 19 500 17 800 16 500 13 OOO 11 500
8.0-11.5. It appears, for example, that missing peak 10 in trace B is actually unresolved and part of peak 11. These results, therefore, suggest differences in the resolution of dissociated forms of these proteins in the two genotypes. The distribution of the proteins across two-dimensional gels is shown in Figure 3. The pattern for Yellow Creole (A) is significantly different from that of Huffman (B). There is a group of proteins with isoelectric points between p H 7.0 and 8.0 around 94 kDa (designated "a" in Yellow Creole) that was not observed in the Huffman assay. In addition, a group of spots a t pH 4.8-6.2 around 14 kDa (area b) in Yellow Creole were not observed in Huffman. In Huffman, a cluster of proteins (area c) at around 30 kDa and pH 7.0-8.5 did not exist in Yellow Creole. The mapping of protein spots in the two genotypes clearly showed variability either in intensity of staining, in the presencelabsence of proteins, or in both of them. Over 50 spots were resolved and detected by visual inspection at
1424 J. Am. FWChem., Vol. 40, No. 8, 1992
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4
proteins that allowfor differentiationof the two genotypes. The results point out that genetic factors responsible for variations in protein amount and presence/absence may be an important basis for recognizing fungal inhibiting proteins that might impart resistance to corn. A comprehensive picture, however, would require protein analyses of a large number of genotypesof corn whose resistance to A. flavus infection has been established in laboratory and/or field studies or analysis of corn progeny in which these proteins and aflatoxin resistance are segregating. LITERATURE CITED
Ly
t3 6.2
6.7
-
0.7
0.4
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Figure 3. Two-dimensionalPAGE of cationicproteins from the two com genotypes [A, Yellow Creole (resistant); B, Huffman (susceptible)]. Isoelectric focusing (IEF) was used in the first dimension and SDS electrophoresis for the second dimension. Numberson the left correspond to the molecularweight markers, and M (arrow) denotes the IEF internal standard, MW 27 OOO. p! = 5.2. The lower case letters a-c denote areas of major differencesin the profiles of the two genotypes. Lines acrossthe gels correspondto specificmolecularweight zones of the markers.
the concentration of protein applied to the isoelectric focusing gels. Insummary,this paperpresenbacartographyofagroup of cationic proteins in kernels of two genotypes of corn that are resistant and susceptible to A. flavus contamination. Both one- and two-dimensionalgel electrophoretic profiles showed the extent of heterogeneity among the
Albersheim,P.; Anderson-Prouty,A. J. Carbohydrates,proteins, cell surfaces, and the biochemistry of pathogenesis. Annu. Rev. Plant Physiol 1975,26,31-52. Barkai-Colan, R.; Mirelman, D.;Sharon, N. Studies on growth inhibition by lectins of Penicillia and Aspergilli. Arch. Microbiol. 1978,116,119-124. Coleman, W.H.;Robert, W. R. Inhibitors of animal cell-free protein synthesis from grains. Biochim. Biophys. Acta 1982, 696,239-244. Laemmli, U. K. Cleavage of atructural proteins during the assembly of the head of bacteriophageT4. Nature 1970,227, 680-685. Landry, J.; Moureaux, T.Distribution and amino acid composition of protein groups located in different histological parts of maize grain. J. Agric. Food Chem. 1980,28,1186-1191. Mikola, J. Disc electrophoretic studies on the basis proteins of barley grain. Ann. Acad. Sci. Fenn., Ser. A2 1965,130,7-47. Neucere, J. N.; Zeringue, H. J., Jr. Inhibition of Aspergillus flavus growth by fractions of salt-extracted proteins from maize kernel. J. Agric. Food Chem. 1987,35,806-808. Oakley, B. R.; Kirsh, D.R.; Morris, R. A simplified silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 1980,105,361-363. OFarrell, P. H. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 1975,250,4007-4021. Olson, V. L.; Hansine, R. L.; McClary,D.0.The role of metabolic energy in the lethal action of basic proteins on Candida albicans. Can. J. Microbiol. 1977,23,166-174. Roberts, W. K.; Selitrennikoff, C. P. Isolation and partial characterization of two antifungal proteins from barley. Biochim. Biophys. Acta 1986,880,161-170. Zuber, M. S.;Darrah,L. L.; Lillehoj, F. B.; Josephson, L. M.; Manwiller, A.; Scott, G. E.; Gudauskas, R. T.; Homer, E. S.; Widstrom, N. W.; Thompson, D. L.; Bockholt, A. J.; Brewbaker, J. L. Comparison of open-pollinated maize varieties and hybrids for prehmest aflatoxin contamination in the Southern United States. Plant Dis. 1983,67,185-187. Received for review September 10,1991. Revised manuscript received February 12,1992. Accepted April 16,1992.
